The control of gene expression depends on DNA-bound proteins and their interactions. This proposal focuses on two well-characterized prokaryotic regulators, lambda repressor and E. coli CAP protein. The long term goal of the proposed research is a detailed molecular understanding of (i) the nature of interactions between regulators bound some distance from one another along DNA, and (ii) the nature of the interaction between an activator and RNA polymerase. Lambda repressor binds cooperatively to pair of operator sites both when those sites are adjacent, as in the natural situation, and in artificial constructs where they are separated. In the latter case, the cooperative interaction induces the formation of a loop in the DNA. The molecular basis of cooperative binding will be genetic screens to identify mutants that exhibit either weaker or stronger cooperativity and by isolating mutant/suppressor pairs. This approach will be complemented by site- directed mutagenesis once specific residues have been implicated. Lambda repressor also stimulates transcription. This activation probably involves a direct contact between DNA=bound repressor and RNA polymerase. The 'activation surface' of repressor is well defined. We propose to use both genetic and biochemical approaches to identify which subunit of polymerase is contacted by repressor and the specific amino acids involved in that interaction. To investigate whether CAP and repressor activate by similar mechanisms the polymerase mutants that are isolated will also be tested for their effect on CAP-stimulated transcription. Two additional issues regarding activation will be addressed: (i) We will examine the basis of the block in activation observed when a repressor bound some distance upstream interacts with the molecule bound at the activation site, (ii) We will determine whether repressor can activate from a distance. Principles derived from analysis of prokaryotic regulatory proteins continue to prove useful in the study of normal and abnormal control of gene expression which underlies many important cellular processes in eukaryotes, including embryogenesis and oncogenesis.